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Applied and Environmental Microbiology, February 2003, p. 812-819, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.812-819.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

Jung-Hoon Bae,1,2 Jung-Hoon Sohn,1 Chang-Seo Park,3 Joon-Shick Rhee,2 and Eui-Sung Choi1*

Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-333,1 Biotech Research Lab, Doosan Corporation Biotech BU, Suji-Eup, Yongin-City, Kyonggi-Do 449-844,3 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong, Taejon 305-701, Korea2

Received 10 September 2002/ Accepted 12 November 2002

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/µg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.

* Corresponding author. Mailing address: Laboratory of Microbial Functions, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-333, Korea. Phone: 82-42-860-4453. Fax: 82-42-860-4594. E-mail: choi4162{at}kribb.re.kr.

Applied and Environmental Microbiology, February 2003, p. 812-819, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.812-819.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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